Presented at the Molecular Interactions in Biopharmaceutical Formulations: Can Stability be Rationalised and Predicted. Held by the Formulation Science and Technology Group of the Royal Society of Chemistry in association with the Academy of Pharmaceutical Sciences.

1. Ozone Detection in Pharmaceutical Containers
Charlotte Bella, Elaine Martina, Brendan Fishb
aBiopharmaceutical and Bioprocessing Technology Centre, Newcastle University, UK
bGlaxoSmithKline, Barnard Castle, UK
1 Introduction charlotte.bell3@ncl.ac.uk 5 Results and Discussion
Pharmaceutical vials are subjected to a high voltage leak detection (HVLD) test. A O O An experiment was performed to determine how long ozone remains in solution,
high voltage (up to 30kV) is passed across the surface of the vial and the presence O3 to understand the maximum time period available between leak testing vials with
N N
of a crack is detected through a change in the current. HVLD has been linked to OH OH
HVLD and testing for the presence of ozone. Deionised water was ozonized and
the breakdown of active pharmaceutical ingredients (APIs) and it is hypothesised HN
NH2
N
O NH2 the presence of ozone was measured using PIT. Figure 4 shows the decomposition
that ozone, a strong oxidising agent, generated as a consequence of the high OHC of ozone over time. After only 20 minutes, the ozone initially present had
voltage, is responsible. (1) It is crucial that the manufacturing process is designed Histidine Histidine oxidation product decomposed, highlighting the need for a method that quickly captures the ozone
to minimise any changes to the molecular structure of biopharmaceuticals and after vials have been subjected to HVLD.
O O
this extends to ensuring that container integrity testing does not cause product O3 O3 O
OH
S 0.25
degradation. S
OH O OH
S
O
O
0.2
NH2 NH2 0 mins
NH2
Absorbance
Methionine Methionine sulfoxide Methionine sulfone 0.15 10 mins
1.4
1.2
Figure 2 – Effect of ozone on histidine and methionine 0.1 20 mins
1
3 Aims Blank Ozonated
Absorbance
0.05 0.8 water
0.6
0
(i) To find out if the HVLD machine causes the formation of ozone inside a vial 500 550 600 650 700 0.4 8mM H2O2
Wavelength (nm)
(ii) How can ozone inside the vial can be quantified? 0.2
Figure 4 – Ozone decomposition over 0
The approach that will be taken is to: time measured using PIT solution 190 220 250 280
Wavelength (nm)
1. Develop an assay for ozone measurement
2. Carry out limit of detection tests Figure 5 – Presence of hydrogen peroxide in
ozonized water confirmed by UV measurement
3. Perform tests on vials subjected to HVLD
4. If ozone is present, look at the impact of ozone on APIs After ozonizing deionised water, the presence of hydrogen peroxide, a
decomposition product of ozone, was detected at a wavelength of ~220nm. Figure
Figure 1 – Ampoule going through HVLD machine 5 shows the UV spectra of ozonized water and of 8mM hydrogen peroxide. After
2 Action of Ozone on Proteins 4 Method Development the development of a standard curve, to relate absorbance to concentration,
hydrogen peroxide could be used as an indicator to detect whether ozone was
present in water, and has decomposed. Testing for hydrogen peroxide may be an
Concern exists surrounding the use of HVLD for biopharmaceuticals due to the The method must be sufficiently sensitive to measure low levels of ozone in the important factor, as it has been previously stated that ,‘the dissociation products
possible degradation caused by the ozone attacking the proteins. Figure 2 shows gaseous and aqueous phases in the vial. of ozone in water may be more powerful oxidization agents than ozone itself’. (7)
the effect of ozone on histidine and methionine. Previous studies (2, 3) have
shown the following: Direct measurement of aqueous ozone using UV absorbance at 258nm was
investigated. By adopting this approach, it was found that the low molar 6 Conclusions, Future Work and Impact
 Ozone attack is generally directed towards aromatic rings absorptivity and fast decomposition of ozone made it difficult to measure.
Vials containing distilled water will be passed through the HVLD machine and then
 Amide bonds of the main protein chain are not degraded by ozone, so no By utilising the Indigo method, the ozone present in aqueous or gaseous samples, the headspace gas and liquid will be tested using the Indigo method. Testing for
significant chain scission occurs and the primary protein structure remains reacts rapidly with potassium indigo trisulfonate (PIT), to form a colourless the presence of hydrogen peroxide may also give an indication that ozone has
intact substance - sulfonated isatin, Figure 3, thereby reducing the absorbance of PIT at been present but has decomposed. If ozone is present in the vial, then the effects
600nm. (4, 5) This method rapidly identifies the presence of ozone prior to its of operational parameters, e.g. voltage, on ozone generation will be investigated.
 Ozone is found to cause changes in secondary and tertiary protein structures, decomposition. A variation of the Beer Lambert law (A=εcℓ) is then used to
due to the partial oxidation of aromatic monomeric units and/or cysteine units calculate the ozone concentration of a sample. Gas tight syringes are used for The composition of vial headspace gas could be changed to an inert gas such as
sampling to improve accuracy and precision (6) compared to previously published nitrogen to avoid ozone generation and hence address possible product safety
 Thiol groups in cysteine form disulphide cross links, causing denaturation and methods that utilise volumetric flasks. issues. The effect of ozone on the long-term stability of protein formulations could
K+
changes in solubility that could lead to precipitation SO3-
O
(SO3-)
be investigated to determine whether protein damage occurs through contact
K+
NH NH with ozone. Currently the HVLD machine is redundant for some products. If these
 Histidine displays reactivity to ozone, due to the presence of an imidazole ring, K+
SO3-
O problems could be mitigated, the HVLD machine could be used to leak test all vials
which is in agreement with the Hospira study (1) when oxidation of the -
O3S -
O3S
filled on the site thus increasing operational efficiency.
HN
imidazole ring in the drug product occurred O O References
(1) McGinley, C. M., et al. Poster: Degradation of Drug Product Caused by a Leak Detection Instrument: Mechanistic Studies of Degradation. Hospira, Inc., Lake Forest, IL 60045.
(2) Cataldo, F. (2006). Ozone Degradation of Biological Macromolecules: Proteins, Hemoglobin, RNA, and DNA. Ozone: Science & Engineering 28(5): 317-328.
Potassium indigo trisulfonate O3 Sulfonated isatin (3) Sharma, V. K. and N. J. D. Graham (2010). Oxidation of Amino Acids, Peptides and Proteins by Ozone: A Review. Ozone: Science & Engineering 32(2): 81-90.
(4) Bader H, Hoigné J (1981) Determination of ozone in water by the indigo method, Water Research 15:449-456.
Figure 3– Conversion of PIT to colourless sulfonated isatin (5) Bader H, Hoigné J (1982) Determination of Ozone In Water By The Indigo Method: A Submitted Standard Method, Ozone: Science & Engineering 4:169-176.
(6) Chiou C-F, Mariñas BJ, Adams JQ (1995) Modified Indigo Method For Gaseous And Aqueous Ozone Analyses, Ozone: Science & Engineering 17:329-344
(7) Peleg, M. (1976). The chemistry of ozone in the treatment of water. Water Research 10(5): 361-365